We propose to answer questions about molecular interactions guiding the earliest steps in formation of infectious cytomegalovirus. Our focus is on two genetically related proteins encoded by open reading frame UL80a, called the assembly protein precursor (pAP) and the capsid maturational protease precursor (pPR). Together these proteins interact with themselves and with other proteins to coordinate assembly and maturation of the nascent procapsid shell - a process essential to produce infectious virus. We have recently overcome a solubility problem that complicates working with the purified proteins in vitro, and will apply this new approach to pursue leads and test models based on encouraging results from pilot studies. We will do this through three Specific Aims: (i) test predicted effects of phosphorylation on key interactions of pAP and pPR; (ii) verify the differential dominance of the carboxyl coiled-coil domain in the self-interaction of pAP versus pPR self-interaction, and determine its biological importance and relevance; and (iii) establish whether a CMV-specific second nuclear localization signal (NLS2) in pAP and pPR is required for their interaction with the capsid portal protein (pUL104). Information gained from this work will help define the sequence of changes that drive UL80 protein associations and dissociations during capsid formation and maturation, and contribute to the longer-term goal of developing new strategies for disrupting (e.g., for therapeutics) or exploiting (e.g., gene/drug delivery vectors) herpesvirus assembly. PUBLIC HEALTH RELEVANCE: Cytomegalovirus is a herpes-group virus that is a threat to people with weakened immune systems, including the very young and the very old. This research seeks to understand how two genetically-related proteins guide the earliest steps in forming infectious virus. Information obtained will relate the molecular interactions driving capsid formation will the biological mechanism of virus formation, and will help identify new ways to interfere therapeutically with that process.